Original Article

Effect of Iron Containing Supplements on Rats' Dental Caries

Progression
AR. Eshghi1
, R. Kowsari-Isfahan2, M. Rezaiefar3, M. Razavi4, S. Zeighami5

1
Assistant Professor, Department of Pediatric Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
2
Postgraduate Student, Department of Pediatric Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
3
Pedodontist, Private Practice
4
Associat Professor, Department of Oral and Maxillofacial Pathology, Isfahan University of Medical Sciences, Isfahan, Iran
5
Dentist, Private Practice


Corresponding author:
AR. Eshghi, Department of
Pediatric Dentistry, Faculty of
Dentistry, Isfahan University of
Medical Sciences, Isfahan, Iran
eshghi@dnt.mui.ac.ir
Received: 19 December 2011
Accepted: 5 February 2012
Abstract
Objective: Iron deficiency is the most common form of malnutrition in develop-
ing countries. Iron containing supplements have been used effectively to solve
this problem. In children, because of teeth staining after taking iron drops, parents
have the idea that iron drops are the cause of tooth decay; therefore, they limit this
vital supplement in their children’s diet. Hereby, we evaluate the histologic effect
of iron containing supplements on tooth caries in rice rats with cariogenic or non-
cariogenic diet.
Materials and Methods: Twelve rats were selected and divided into four groups
for this interventional experimental study. Four different types of dietary regi-
mens were used for four months; group A, cariogenic diet with iron containing
supplements; group B, cariogenic diet without iron containing supplements; group
C, non-cariogenic diet with iron containing supplements; group D, non-cariogenic
diet without iron containing supplements. After sacrificing the rats, 20-micron
histological sections of their posterior teeth were prepared using the Ground Sec-
tion method, then they were studied under polarized light microscopy. In order to
compare the progression of caries in different samples, the depth of the lesions in
the enamel was measured as three grades I, II and III.
Results: The mean grade value of A, B, C and D groups were 1.61, 2.61, 1.37
and 1.80, respectively. Statistical analysis revealed that significantly fewer caries
were seen in the group which had received iron containing supplements and cari-
ogenic diet compared with cariogenic diet without iron supplements (p<0.05).
Conclusion: Ferrous sulfate reduces the progression of dental caries in the cari-
ogenic dietary regimen.
Key Words: Iron; Dental Caries; Dietary Regimen; Rat
Journal of Dentistry, Tehran University of Medical Sciences, Tehran, Iran (2012; Vol. 9, No.1)

INTRODUCTION stances, this deficiency is alleviated by sup-

Iron deficiency is the most widely distributed plementing various foods with iron salts [3].
nutrient deficiency state, affecting more than 2 On the other hand, iron supplementation for
billion people worldwide [1,2]. In some in- children under 5-years old is recommended on

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Journal of Dentistry, Tehran University of Medical Sciences
the basis of anemia prevalence [1].
Caries is a diet-dependent disease, the preva-
lence of which may be reduced by avoiding
frequent ingestion of sugars [4]. An alternative
strategy would be to reduce the cariogenicity
of sucrose through appropriate supplementa-
tion [5].
In countries where iron deficiency is apparent,
the prevalence of dental caries is high, even
though the two phenomena are not necessarily
directly related [3].
Results from studies conducted in vitro, in situ
and in vivo, both in humans and animals, sup-
port the belief that iron may have cariostatic
properties [6].
Pecharki et al suggested that iron ions may re-
duce in situ the cariogenic potential of sucrose
and the effect seems to be related to the reduc-
tion of Streptococci mutans in the dental bio-
film formed [5].
Miguel et al in two studies done in desalivated
rats found that iron added to sucrose, alone or
in combination with other ions has a great ef-
fect on the reduction of the carigenic potential
of the sugar [3,7].
In addition Devulapalle and Mooser showed
the inhibition of the activity of glucosyltrans-
ferase by ferrous sulfate in vitro [8]. Recently,
it has been shown that iron can inhibit acid
demineralization by directly affecting mineral
dissolution [9,10-13].
Polarized light microscopy has been used to
explore the histologic progression of caries
lesions.
Microscopic inspection is based on optical
changes in the tissues. For the enamel, this is
fairly straightforward, since a reduced mineral
content is readily observed as opacity [14].
Knowing the fact that pediatricians prescribe
iron supplements for children routinely, this
study was designed to histologically evaluate
the effect of an iron containing supplement
(ferrous sulfate oral drops, Daropakhsh, Iran)
on tooth caries in rice rats either with cari-
ogenic or non-cariogenic diet.
2 15
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Eshghi et. al
MATERIALS AND METHODS
In this study, twelve 21-day-old weaning rice
rats of both genders were selected. The ani-
mals were maintained under standard laborato-
ry conditions and divided into four groups ac-
cording to their diet (A, B, C and D). As each
rat has twelve posterior teeth, 36 teeth were
studied in each group. The dietary regimen
was specified for each group as:
Group A: Cariogenic diet with iron containing
supplement. Group B: Cariogenic diet without
iron containing supplement. Group C: Non-
cariogenic diet with iron containing supple-
ment and Group D: Non-cariogenic diet with-
out iron containing supplement.
The difference between cariogenic and non-
cariogenic diet in this study was the sucrose
sweetened water (5% w/v) added to a com-
mercial laboratory diet (non-cariogenic).
The iron containing supplement “ferrous sul-
fate oral drop which contains 125 mg/ml iron”
(Daroopakhsh, Tehran, Iran) was admini-
strated as five drops each time, three times a
week. All groups were infected with Strepto-
coccus sobrinus which had been passaged
through a desalivated rat to enhance virulence
to obtain the desirable cariogenic condition
[3].
After four months, the rats were sacrificed by
chloroform solution and the mandible and
maxilla were removed, washed in running wa-
ter and placed in 10% formaldehyde for 24
hours. The pieces were washed, dried with
gauze and dissected. Each hemi jaw was sec-
tioned along the sagittal plane. Ground Section
Method was used for preparing histological
sections. Longitudinal sections of 20 microns
were prepared in a manner that the central
grooves were perfectly exposed (Fig 1). After
preparing, the samples were studied by one
pathologist using polarized light microscopy.
As cariogenesis in enamel causes a disruption
in the arrangement of hydroxyapatite crystals,
discolored areas with irregularities in the later-
al joints to the central grooves were observed
2012; Vol. 9, No. 1
Eshghi et. al
Fig 1. A schematic presentation of the main cen-
tral fissure of a rat’s mandibular second molar.
Abbreviations: D, dentin; E, enamel; F, fissure; P,
pulp tissue; PD, predentin layer
under polarized light. In order to compare
these areas in different samples, based on the
depth of caries in the enamel, an arbitrary qua-
litative classification in three different grades
was established; grade I: A discolored area
extending from the surface to the first one-
third of the distance from the central groove
surface to the dentin surface; grade II: A disco-
lored area extending from the surface to the
first two-thirds of the distance from the central
groove surface to the dentine surface; grade
III: A discolored area extending from the sur-
face over the first two-thirds of the distance
from the central groove surface to the dentine
surface.
To examine the reliability of measurements,
20 samples were chosen randomly and caries
grading was performed a second time after a
long time interval, and a high correlation of
repeated measurements was found (r = 0.91).
The teeth in a rat mouth could not be analyzed
independently, so the mean value obtained
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Effect of Iron Containing Supplements on Rats' Dental…
from one rat (three mean values for each
group) were analyzed as the data using
ANOVA test. SPSS software (version 11.5)
was used and the significance limit was set at
5%.
RESULTS
All animals maintained good health during the
experimental period.
But in the preparation of the sections of the
teeth some of them were missed. It is neces-
sary to mention that due to the rather sustained
exposure to cariogenic bacteria, no sample was
observed without any decay.
There was no dentinal lesion among the
groups and for the enamel lesion the following
results were obtained:
group A, which had received iron supplement
with cariogenic diets, provided a total number
of 17 teeth.
Microscopic examinations revealed that 10
caries were grade I, four were grade II, and the
decay in three samples was of grade III (mean
grade value, 1.61).
Group B, which had received cariogenic diet
yielded 17 teeth out of which only one sample
was grade I, while four were grade II and 12
samples had caries of grade III (mean grade
value, 2.61).
In group C, which had received iron contain-
ing supplement with non-cariogenic diets, of
the total 24 teeth which were maintained
sound in preparation, 15 samples were grade I,
nine were grade II and none showed grade III
(mean grade value, 1.37).
Group D, which had received only non-
cariogenic diet without any supplement, pro-
vided 21 teeth for the study, six samples were
grade I, 11 were grade 2 and four were grade
III (mean grade value, 1.80) (Table 1).
Statistical analysis revealed that significantly
fewer caries were only seen in groups which
had received iron containing supplement and
cariogenic diet compared with cariogenic diet
without iron supplement (p<0.05).
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DISCUSSION by using iron in sucrose and experimental diet

The purpose of this study was to evaluate the
effect of iron containing supplements on rats'
dental caries progression and we found that
such supplements might have an inhibitory
role on progression of dental caries when us-
ing the cariogenic regimen. Even though using
a non-cariogenic diet could reduce the depth of
the enamel lesion, it could not prevent caries
occurrence. Iron containing supplements could
not significantly inhibit progression of caries
in the non-cariogenic type of regimen.
Our results confirmed the results of the pre-
vious studies and agreed with the inhibitory
effect of iron ion in the progression of dental
caries.
Although missing teeth in the section prepara-
tion procedure was a limitation of our study,
the total number of samples compromises it.
The relevance of data obtained from numbers
of teeth in one rat’s mouth was the other limi-
tation.
Several investigations have previously offered
the inhibitory effect of iron on caries devel-
opment. Some studies evaluated this effect
suggesting the inhibitory role of iron in dental
caries progression [3,5,7,15]. Results from our
study confirmed these findings only in the ca-
riogenic type of regimen, but in the non-
cariogenic regimen, the inhibitory effect of
iron was not significant. Maybe more speci-
mens were needed to make it. Some others
studied the effect of iron on acid demineraliza-
tion of the enamel in vitro. They also found
that a specific concentration of iron could re-
duce acid demineralization of the enamel
[6,10-13].
Another study traced iron ions in human and
rice rats' teeth using X-ray fluorescent. They
observed that teeth with fewer caries had high-
er pigment concentration indicating that they
received more iron [16].
Although the results of these studies indicated
the inhibitory effect of iron absorption on cari-
ogenesis, none of them had a controlled diet or
used histological observations.
The present study obtained the same results,
but in a solid manner in which the specific die-
tary regimen used in different groups during

Table 1. Distribution of Caries in Four Groups with Different Diets

Grade I Grade II Grade III

Group Coronal one Mean Grade value

Coronal two
(Whole enamel or beyond it)
third of enamel third of enamel

A 10 4 3 1.61

B 1 4 12 2.61

C 15 9 0 1.37

D 6 11 4 1.80
Groups: A, Cariogenic diet with iron supplement B, Cariogenic diet without any supplement C, non-Cariogenic
diet with iron supplement D, Non-cariogenic diet without any supplement

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Eshghi et. al
the investigation period and samples was his-
tologically evaluated under polarized light mi-
croscope (a more accurate method), leading to
a higher level of reliability for results. Based
on our results, it may be concluded that al-
though oral iron supplements may cause teeth
discoloration due to the formation of iron salts
on teeth surfaces [15,17], it plays an inhibitory
role in progression of dental caries. This inhi-
bitory role seems to be due to increased ena-
mel resistance, resulting from the protection
by iron salts against acidic productions of oral
bacteria [3,6,10-13] and the reduction in the
population of Streptococci mutans in the den-
tal biofilm formed [5]. However, the inactiva-
tion of oral bacterial glucosyltransferase by
metallic ions such as iron should be mentioned
[3,8].
CONCLUSION
In conclusion, it is suggested that when iron
containing supplements are used, despite some
side effects such as teeth discoloration, reduc-
tion of dental caries progression is expected
when the cariogenic dietary regimen is used.
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